Wakefulness, endocrine status, and metabolism are strongly influenced by circadian transcriptional clocks present in most nucleated cells of the body. A core clock transcriptional factor, BMAL1, is in turn regulated by several orphan members of the nuclear receptor (NR) family through a conserved promoter region. Two of these receptors have been crystallized with naturally-occurring, exchangeable lipids in their ligand-binding domains (LBDs) The LBDs have structural features typical of other NRs, such as the steroid, retinoid, or thyroid receptors, providing compelling evidence that the receptors function as small molecule ligand responsive transcription factors. Pharmacologically active ligands to these two closely- related orphan NRs have yet to be identified, however. Successful ligand discovery at the two receptors will have a major impact on research in mammalian circadian biology and will facilitate development of novel small molecule therapeutics for sleep and mood disorders, for diabetes and other forms of metabolic disease, and for cancer. The first objective of this proposal is to implement a homogenous, solution phase fluorescence resonance energy transfer (FRET) assay for these two targets in a 384-well format. The biochemical components of the assay have been identified and tested. The assay should respond to both agonists and antagonists. A library of 25,000 compounds will be screened and hits confirmed by a secondary assay in order to find positive control ligands for assay validation and optimization for HTS. If necessary, hits will be modified by medicinal chemistry synthesis to achieve adequate potency (EC50 < 5 fM) for confirmatory assays. These findings, and migration of a proven assay to a dedicated HTS facility, have the potential to considerably accelerate ligand discovery at these targets. Relevance to Public Health Human physiology, in health and disease, is heavily influenced by the autonomous daily cycle that regulates all aspects of body function. The findings that emerge from these studies have to potential to address these processes for the benefit of human health.